Cytotoxic T cell epitope peptide for SARS coronavirus, and use thereof

ABSTRACT

The present invention aims to provide a novel CTL epitope peptide of the SARS coronavirus. The present invention provides a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/JP2009/070043 filed Nov. 27, 2009, claiming priority based onJapanese Patent Application No. 2008-304965, filed Nov. 28, 2008, thecontents of all of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a cytotoxic T cell epitope peptide forthe SARS coronavirus and use thereof.

BACKGROUND ART

Severe Acute Respiratory Syndrome (SARS) is an emerging infectiousdisease with a high lethality caused by the novel SARS coronavirus(SARS-CoV). Since the outbreak of the disease in 2003 in China, not lessthan 8000 people have been infected and about 800 people have died.However, no effective prophylactic or therapeutic method for the diseaseexists so far. After the outbreak of SARS, the SARS virus, which is thecausative virus of SARS, was identified (Non-patent Document 1), and itsbase sequence has been determined (Non-patent Document 2).

The SARS coronavirus is a novel species of coronavirus belonging toCoronaviridae, a group of single-stranded (+) RNA viruses. The genomesize of the SARS coronavirus is 29.7 kb, which is very large (Non-patentDocument 2), and the genome encodes 23 putative proteins. As majorstructural proteins, there are Spike (1256 aa), Nucleocapsid (423 aa),Membrane (222 aa) and Small Envelope (77 aa). As nonstructural proteins,there are two polyproteins pp1a (4382 aa, SEQ ID NO:31; GenBankAccession No. AAP13439) and pp1b (2696 aa), and from these polyproteins,individual proteins are cleaved out by proteases in a site-specificmanner.

CITATION LIST Non Patent Literature

-   Non-patent Document 1: Ksiazek, T. G. et al., N. Engl. J. Med.    348:1953-1966 (2003)-   Non-patent Document 2: Marra, M. A. et al., Science 300:1399-1404    (2003)-   Non-patent Document 3: Yang, Z. Y. et al., Nature 428:561-564 (2004)-   Non-patent Document 4: Wang. Y. D. et al., J. Virol. 78:5612-5618    (2004)-   Non-patent Document 5: Chen, H. et al., J. Immunol. 175:591-598    (2005)-   Non-patent Document 6: Zhou, M. et al., J. Immunol. 177:2138-2145    (2006)

SUMMARY OF INVENTION Technical Problem

It has been reported so far that a virus-neutralizing antibody isinduced by a DNA vaccine encoding the Spike protein (Non-patent Document3). Although humoral immunity and cell-mediated immunity are necessaryfor effective defense reaction, cell-mediated immunity has been lessstudied compared to humoral immunity in the field of the SARScoronavirus. Cytotoxic T lymphocytes (CTLs) play an important role forprotection against viruses in cell-mediated immunity, and therefore anew therapeutic method may be provided by controlling the CTL activityspecific to the SARS coronavirus. From this viewpoint, identification ofa strong CTL epitope peptide having a high antigenicity among the SARScoronavirus proteins has been demanded. Several types of partialpeptides derived from the Spike protein, which is a structural protein,have been identified so far as CTL epitope peptides specific to the SARScoronavirus (Non-patent Documents 4 to 6). However, CTL epitope peptidesderived from nonstructural proteins have not been reported yet.

The present invention aims to provide a novel CTL epitope peptide of theSARS coronavirus. More particularly, the present invention aims toprovide a peptide comprising a novel CTL epitope derived from the SARScoronavirus pp1a protein, a peptide-bound liposome and an antigenpresenting cell. Furthermore, the present invention aims to provide aninducing agent for HLA-A2-restricted CTLs, which comprises the peptide,the peptide-bound liposome or the antigen presenting cell as an activeingredient and is specific to the SARS coronavirus. The presentinvention also aims to provide a vaccine and the like for therapy orprophylaxis of infection by the SARS coronavirus.

Solution to Problem

The present inventors carried out the following studies in order toidentify a CTL epitope derived from the SARS coronavirus, which has notbeen reported so far. First, by focusing on the pp1a protein, which is anonstructural protein of the SARS coronavirus, 30 kinds of peptides wereselected among peptides predicted as candidates of the epitope.Subsequently, the binding affinity to the HLA-A2 molecule, which is aMHC class I molecule, was confirmed for the peptides, and 9 kinds ofpeptides among the epitope candidate peptides were found to havesignificant CTL inducing activities. Furthermore, it was revealed that,by immunization with peptide-bound liposomes containing the peptides,the CTL induction is initiated and the CTL response is activated invivo. Based on these findings, the present inventors succeeded inproviding peptides comprising novel CTL epitopes derived from the SARScoronavirus pp1a protein, thereby completing the present invention.

That is, the present invention provides a peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 10, 11,12, 13, 15, 17, 18, 23 and 24. Furthermore, the peptide of the presentinvention preferably comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 12, 15, 17 and 24, and morepreferably comprises the amino acid sequence shown in SEQ ID NO:24. Thepeptide of the present invention is characterized in that it comprises acytotoxic T cell epitope specific to the SARS coronavirus, and that itcomprises an HLA-A2-restricted cytotoxic T cell epitope.

The present invention provides a peptide-bound liposome, wherein apeptide is bound to the surface of a liposome, wherein the liposomecomprises a phospholipid comprising a C₁₄-C₂₄ acyl group containing oneunsaturated bond, or a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond, and a stabilizer, and wherein the peptide is at leastone peptide selected from the above-described peptide.

The above-described phospholipid preferably comprises a C₁₄-C₂₄ acylgroup containing one unsaturated bond, and more preferably comprising anoleoyl group. Furthermore, the above phospholipid is preferably at leastone selected from the group consisting of diacylphosphatidylserine,diacylphosphatidylglycerol, diacylphosphatidic acid,diacylphosphatidylcholine, diacylphosphatidylethanolamine,succinimidyl-diacylphosphatidylethanolamine andmaleimide-diacylphosphatidylethanolamine.

If the phospholipid comprised in the liposome has such a constitution,CTLs for killing pathogen-infected cells can be efficiently enhanced,and prophylaxis and therapy of infectious diseases becomes possible.

The above-described stabilizer is preferably a cholesterol. By thisconstitution, the above liposome can be more stabilized.

The above-described peptide is preferably bound to the phospholipidcomprised in the liposome. By this way, the peptide can be presented onthe surface of the liposome, thereby CTLs specific to the SARScoronavirus can be induced more effectively.

Furthermore, the peptide-bound liposome of the present inventionpreferably comprises the following constituents:

(A) 1 to 99.8 mol % of a phospholipid comprising a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond; and

(B) 0.2 to 75 mol % of a stabilizer.

Furthermore, the peptide-bound liposome of the present inventionpreferably comprises the following constituents:

(I) 1 to 85 mol % of an acidic phospholipid comprising a C₁₄-C₂₄ acylgroup containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond;

(II) 0.01 to 80 mol % of a neutral phospholipid comprising a C₁₄-C₂₄acyl group containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbongroup containing one unsaturated bond, at a concentration of;

(III) 0.2 to 80 mol % of a phospholipid comprising a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond, wherein the phospholipid is bound to atleast one peptide selected from the above-described peptide; and

(IV) 0.2 to 75 mol % of a stabilizer.

The present invention provides an antigen presenting cell prepared bycontacting a cell expressing a cell surface antigen HLA-A2 with at leastone peptide selected from the above-described peptide in vitro. The cellis preferably autologous, and more preferably allogenic.

Furthermore, the present invention provides an inducing agent forHLA-A2-restricted CTLs specific to the SARS coronavirus comprising atleast one peptide selected from the above-described peptide, thepeptide-bound liposome or the antigen presenting cell as an activeingredient.

The present invention provides a vaccine for prophylaxis of infection bythe SARS coronavirus comprising at least one peptide selected from theabove-described peptide, the peptide-bound liposome or the antigenpresenting cell as an active ingredient.

Furthermore, the present invention provides a method for providingimmunity to a subject who needs to be given immunity against the SARScoronavirus comprising administering at least one peptide selected fromthe above-described peptide, the peptide-bound liposome or the antigenpresenting cell to the subject.

The present invention provides a method for providing immunity to asubject who needs to be given immunity against the SARS coronaviruscomprising: collecting cells from the subject; preparing antigenpresenting cells by contacting the cells with at least one peptideselected from the above-described peptide in vitro; and reinjecting theantigen presenting cells to the subject. The cells are preferablylymphoid monocytes, and more preferably dendritic cells.

Advantageous Effects of Invention

The present invention provides a peptide comprising a novel CTL epitopederived from the SARS coronavirus pp1a protein, a peptide-bound liposomeand an antigen presenting cell. Since the CTL induction is initiated andthe CTL response is activated in vivo by the peptide, the peptide-boundliposome or the antigen presenting cell, any of the peptide, thepeptide-bound liposome and the antigen presenting cell of the presentinvention can be used as a CTL inducing agent and/or a vaccine forelimination of the SARS coronavirus. Furthermore, since nonstructuralproteins are synthesized earlier than structural proteins during theprocess of virus infection, a virus elimination effect at the initialstage of virus infection can be expected by activating the immunereaction using a CTL epitope derived from the pp1a protein, which is anonstructural protein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows staining plots for CD8 and IFN-γ by flow cytometry in miceimmunized with a peptide-bound liposome. In FIG. 1, the legends “No pep”and “with pep” each indicate that the immunization was conducted using aliposome alone and a peptide-bound liposome, respectively. In FIG. 1,the legends “Lip-pp1a-xxxx” each indicate a peptide-bound liposomewherein the peptide is a SARS coronavirus pp1a epitope having a sequenceshown in Table 1.

FIG. 2 shows staining plots for CD8 and IFN-γ by flow cytometry in miceimmunized with a spike-1203-peptide-bound liposome. In FIG. 2, thelegends “No pep” and “with pep” each indicate that the immunization wasconducted using a liposome alone and a peptide-bound liposome,respectively. In FIG. 2, the legend “Lip-SARS Spike 1203” indicates apeptide-bound liposome wherein the peptide is a SARS Spike 1203 epitopeof SEQ ID NO: 34 (FIAGLIAIV).

FIG. 3 shows staining plots for CFSE (carboxy fluorescein diacetatesuccinimidyl ester) by flow cytometry in mice which werebooster-immunized with CFSE-labeled antigen presenting cells. In FIG. 3,the legends “No pep” and “with pep” each indicate that the immunizationwas conducted using a liposome alone and a peptide-bound liposome,respectively. In FIG. 3, the legends “Lip-pp1a-xxxx” each indicate apeptide-bound liposome wherein the peptide is a SARS coronavirus pp1aepitope having a sequence shown in Table 1.

DESCRIPTION OF EMBODIMENTS

The best mode for carrying out the invention will be described in detailbelow.

The peptide in the present invention comprises a CTL epitope derivedfrom the SARS coronavirus pp1a protein, and particular examples thereofinclude peptides containing an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24,and peptides comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24.Furthermore, an amino acid sequence selected from the group consistingof SEQ ID NOs: 10, 12, 15, 17 and 24 is preferred, and the amino acidsequence shown in SEQ ID NO:24 is more preferred. These peptidescomprise an HLA-A2-restricted CTL epitope, more particularly, anHLA-A*0201-restricted CTL epitope. The peptide of the present inventionmay be used in various forms (e.g., unmodified, fusion, glycosylated andnonglycosylated), and may contain a C-terminal modification (amidation,esterification, modification with aldehyde or the like), N-terminalmodification (acetylation, biotinylation, fluorescent labeling or thelike) and chemical modification of a functional group (phosphorylation,sulfation, biotinylation or the like).

The peptide may be synthesized according to a method used inconventional peptide chemistry. Examples of the known method of peptidesynthesis include those described in literatures (Peptide Synthesis,Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc.,New York, 1976; Peptide Synthesis, MARUZEN Co., Ltd., 1975; Fundamentalsand Experiments of Peptide Synthesis, MARUZEN Co., Ltd., 1985;Development of Pharmaceuticals, Continued Edition, Vol. 14, PeptideSynthesis, Hirokawa Shoten, 1991) and the like.

The peptide of the present invention as described above may be usedsolely or in combination of plural types of peptides as an inducingagent for HLA-A2-restricted CTLs specific to the SARS coronavirus and/oras a vaccine for therapy or prophylaxis of infection of the SARScoronavirus. That is, since the peptide of the present invention canbind to a HLA-A2 molecule and is presented by cells that express theHLA-A2 molecule, thereby strongly induce CTLs, the peptide of thepresent invention can be used as a CTL inducing agent and/or a vaccinefor elimination of the SARS coronavirus.

The liposome used for the peptide-bound liposome of the presentinvention is a phospholipid bilayer membrane having a closed space.

The liposome used for the peptide-bound liposome of the presentinvention comprises: a phospholipid comprising a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond; and a stabilizer. A phospholipidcomprising a C₁₄-C₂₄ acyl group containing one unsaturated bond ispreferably used as the phospholipid.

The carbon number of the acyl group in the phospholipid comprising aC₁₄-C₂₄ acyl group containing one unsaturated bond is preferably 16 to22, more preferably 18 to 22, and most preferably 18. Particularexamples of the acyl group include palmitoleoyl, oleoyl and erucoyl, andmost preferably oleoyl. The carbon number of the hydrocarbon group inthe phospholipid comprising a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond is preferably 16 to 22, more preferably 18 to 22, andmost preferably 18. Particular examples of the hydrocarbon group includetetradecenyl, hexadecenyl, octadecenyl, C₂₀ monoene, C₂₂ monoene and C₂₄monoene and the like. The unsaturated acyl groups or unsaturatedhydrocarbon groups bound to the 1-position and the 2-position of theglycerin residue comprised in the phospholipid may be either the same ordifferent. In view of industrial productivity, the groups at the1-position and the 2-position are preferably the same.

In view of enhancement of the CTL activity to a practically sufficientlevel, the phospholipid preferably comprises a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond. In cases where the carbon number of theacyl group is less than 13, the liposome may be unstable, or the CTLactivity enhancement effect may be insufficient. In cases where thecarbon number of the acyl group is more than 24, the liposome may beunstable.

Examples of the phospholipid comprising a C₁₄-C₂₄ acyl group containingone unsaturated bond, or comprising a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond include acidic phospholipids, neutralphospholipids, and reactive phospholipids comprising a functional groupto which a peptide can be bound. Their types and ratios may be selecteddepending on various demands.

Examples of the acidic phospholipids which may be used includephosphatidylserine, phosphatidylglycerol, phosphatidic acid andphosphatidylinositol. In view of enhancement of the CTL activity to apractically sufficient level, industrial supply capacity, quality foruse as a pharmaceutical, and the like, diacylphosphatidylserine,diacylphosphatidylglycerol, diacylphosphatidic acid anddiacylphosphatidylinositol comprising a C₁₄-C₂₄ acyl group containingone unsaturated bond are preferably used. Since an acidic phospholipidgives an anionic group on the surface of a liposome, a negative zetapotential is given on the surface of the liposome. Therefore, theliposome gains charge repulsion and can exist as a stable formulation inan aqueous solvent. Thus, an acidic phospholipid is important in view ofensuring the stability of the liposome in an aqueous solvent.

Examples of the neutral phospholipids include phosphatidylcholine. Theneutral phospholipids which may be employed in the present invention maybe used by selecting their types and amounts appropriately within theranges in which enhancement of the CTL activity can be achieved.Compared to an acidic phospholipid and a phospholipid to which a peptideis bound, a neutral phospholipid has a higher function to stabilize aliposome and hence can enhance the stability of the membrane. From thisviewpoint, the liposome to be used for the peptide-bound liposome of thepresent invention preferably contains a neutral phospholipid. The amountof the neutral phospholipid to be used can be determined after securingthe contents of the acidic phospholipid for achievement of the CTLactivity enhancement effect, the reactive phospholipid for the peptidebond and the stabilizer.

The peptide of the present invention is bound to the surface of theliposome by being bound to a phospholipid comprising a C₁₄-C₂₄ acylgroup containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond, wherein the phospholipid is comprisedin the liposome. As the phospholipid for such binding of the peptide, areactive phospholipid comprising a functional group to which the peptidecan be bound is used. The type and the amount of the reactivephospholipid comprising a C₁₄-C₂₄ acyl group containing one unsaturatedbond, or comprising a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond are appropriately selected depending on variousdemands. Similarly to the case of the above-described phospholipid, itis also not preferred that the carbon number of the unsaturated acylgroup or the unsaturated hydrocarbon group comprised in the phospholipidis more than 24 or less than 14 in the case of the reactivephospholipid.

Examples of the reactive phospholipid include phosphatidylethanolamineand terminally modified derivatives thereof. Furthermore,phosphatidylglycerol, phosphatidylserine, phosphatidic acid andphosphatidylinositol, and terminally modified derivatives thereof mayalso be used as the reactive phospholipid. In view of industrialavailability, simplicity of the process of binding with a peptide, yieldand the like, phosphatidylethanolamine or a terminally modifiedderivatives thereof is preferably used. Phosphatidylethanolamine has anamino group to which an antibody can be bound at its terminus.Furthermore, in view of enhancement of the CTL activity to a practicallysufficient level, stability in a liposome, industrial supply capacity,quality for use as a pharmaceutical, and the like,diacylphosphatidylethanolamine comprising a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond or a terminally modified derivativesthereof is most preferably used.

Diacylphosphatidylethanolamine can be obtained by, for example, usingdiacylphosphatidylcholine as a crude material and carrying out abase-exchange reaction of choline and ethanolamine using phospholipaseD. More particularly, a solution of diacylphosphatidylcholine inchloroform is mixed at an appropriate ratio with water in whichphospholipase D and ethanolamine are dissolved, to obtain a crudereaction product. The crude reaction product is purified with a silicagel column using a chloroform/methanol/water solvent, thereby obtainingthe diacylphosphatidylethanolamine of interest. Those skilled in the artcan carry out the purification by appropriately selecting the conditionsfor purification by the column, such as the composition ratio of thesolvent.

Examples of the terminally modified derivatives include a terminallymodified diacylphosphatidylethanolamine produced by binding one of thetermini of a divalent reactive compound to the amino group ofdiacylphosphatidylethanolamine. Examples of the divalent reactivecompound which may be used include compounds comprising, at least oneterminus, an aldehyde group or a succinimide group which can react withthe amino group of diacylphosphatidylethanolamine. Examples of thedivalent reactive compound comprising an aldehyde group include glyoxal,glutaraldehyde, succindialdehyde and terephthalaldehyde. Preferredexamples thereof include glutaraldehyde. Examples of the divalentreactive compound comprising a succinimide group includedithiobis(succinimidylpropionate), ethyleneglycol-bis(succinimidylsuccinate), disuccinimidyl succinate,disuccinimidyl suberate and disuccinimidyl glutarate.

Furthermore, examples of the divalent reactive compound comprising asuccinimide group at one terminus and a maleimide group at the otherterminus include N-succinimidyl4-(p-maleimidophenyl)butyrate,sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate,N-succinimidyl-4-(p-maleimidophenyl)acetate,N-succinimidyl-4-(p-maleimidophenyl)propionate,succinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate,sulfosuccinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate,N-(γ-maleimidobutyryloxy)succinimide andN-(ε-maleimidocaproyloxy)succinimide. By using such a divalent reactivecompound, a terminally modified diacylphosphatidylethanolaminecomprising a maleimide group as a functional group is obtained. Bybinding a functional group at one terminus of such a divalent reactivecompound to the amino group of diacylphosphatidylethanolamine, aterminally modified diacylphosphatidylethanolamine can be obtained.

Examples of the method for binding the peptide to the surface of theliposome include a method wherein a liposome comprising the abovereactive phospholipid is prepared and the peptide is then added theretoto bind the peptide to the reactive phospholipid in the liposome.Furthermore, by preliminarily binding the peptide to the reactivephospholipid and mixing the obtained peptide-bound reactive phospholipidwith a phospholipid other than a reactive phospholipid and a stabilizer,a liposome in which the peptide is bound to its surface can also beobtained. The method for binding a peptide to a reactive phospholipid iswell-known in the art.

The liposome to be used for the peptide-bound liposome of the presentinvention comprises at least 1 type, for example, 2 or more types,preferably 3 or more types of phospholipid(s) comprising a C₁₄-C₂₄ acylgroup containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond. For example, the liposome to be used inthe peptide-bound liposome of the present invention comprises at least 1type, for example, 2 or more types, preferably 3 or more types ofphospholipid(s) comprising a C₁₄-C₂₄ acyl group containing oneunsaturated bond, or a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond selected from diacylphosphatidylserine,diacylphosphatidylglycerol, diacylphosphatidic acid,diacylphosphatidylcholine, diacylphosphatidylethanolamine,succinimidyl-diacylphosphatidylethanolamine andmaleimide-diacylphosphatidylethanolamine.

Furthermore, the liposome to be used for the peptide-bound liposome ofthe present invention preferably comprises at least one type of each of:

acidic phospholipid comprising a C₁₄-C₂₄ acyl group containing oneunsaturated bond, or a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond;

neutral phospholipid comprising a C₁₄-C₂₄ acyl group containing oneunsaturated bond, or a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond; and

reactive phospholipid comprising a C₁₄-C₂₄ acyl group containing oneunsaturated bond, or a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond.

In the present invention, sterols and tocopherols may be used as thestabilizer of the liposome. The sterols may be those generally known assterols, and examples thereof include cholesterol, sitosterol,campesterol, stigmasterol and brassicasterol. In view of availabilityand the like, cholesterol is especially preferably used. The tocopherolsmay be those generally known as tocopherols, and preferred examplesthereof include commercially available α-tocopherol in view ofavailability and the like.

Furthermore, as long as the effect of the present invention is notadversely affected, the liposome to be used for the peptide-boundliposome of the present invention may contain known liposomeconstituting components that can constitute a liposome.

Examples of the composition of the liposome to be used for thepeptide-bound liposome of the present invention include the following:

(A) 1 to 99.8 mol % of a phospholipid comprising: a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond; and

(B) 0.2 to 75 mol % of a stabilizer.

The content of each component is represented as mol % with respect tothe total constituting components of the peptide-bound liposome.

In view of the stability of the liposome, the content of the component(A) is preferably 10 to 90 mol %, more preferably 30 to 80 mol %, stillmore preferably 50 to 70 mol %.

In view of the stability of the liposome, the content of the component(B) is preferably 5 to 70 mol %, more preferably 10 to 60 mol %, stillmore preferably 20 to 50 mol %. In cases where the content of thestabilizer is more than 75 mol %, the stability of the liposome isdeteriorated, which is not preferred.

The component (A) comprises the followings:

(a) a phospholipid, to which a peptide is not bound, comprising aC₁₄-C₂₄ acyl group containing one unsaturated bond, or a C₁₄-C₂₄hydrocarbon group containing one unsaturated bond; and

(b) a phospholipid, to which a peptide is bound, comprising a C₁₄-C₂₄acyl group containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbongroup containing one unsaturated bond.

The content of the component (a) is usually 0.01 to 85 mol %, preferably0.1 to 80 mol %, more preferably 0.1 to 60 mol %, still more preferably0.1 to 50 mol %.

The content of the component (b) is usually 0.2 to 80 mol %, preferably0.3 to 60 mol %, more preferably 0.4 to 50 mol %, still more preferably0.5 to 25 mol %. In cases where the content is less than 0.2 mol %, theamount of the peptide becomes low, and therefore it is difficult toactivate CTLs to a practically sufficient level. In cases where thecontent is more than 80 mol %, the stability of the liposome becomeslow.

The phospholipid of the component (a) usually includes theabove-mentioned acidic phospholipid and neutral phospholipid. Thephospholipid of the component (b) includes the above-mentioned reactivephospholipid.

The content of the acidic phospholipid is usually 1 to 85 mol %,preferably 2 to 80 mol %, more preferably 4 to 60 mol %, still morepreferably 5 to 40 mol %. In cases where the content is less than 1 mol%, the zeta potential becomes small and the stability of the liposomebecomes low, and it is difficult to activate CTLs to a practicallysufficient level. On the other hand, in cases where the content is morethan 85%, the content of the peptide-bound phospholipid in the liposomebecomes low as a result, and therefore it is difficult to activate CTLsto a practically sufficient level.

The content of the neutral phospholipid is usually 0.01 to 80 mol %,preferably 0.1 to 70 mol %, more preferably 0.1 to 60 mol %, still morepreferably 0.1 to 50 mol %. In cases where the content is more than 80.0mol %, the contents of the acidic phospholipid, the peptide-boundphospholipid and the liposome stabilizer comprised in the liposomebecome low, and it is difficult to activate CTLs to a practicallysufficient level.

The peptide-bound phospholipid is obtained by binding a peptide to thereactive phospholipid, and the ratio of binding of the reactivephospholipid to the peptide may be selected within the range in whichthe effect of the present invention is not adversely affected, by takingthe type of the functional group used in the binding, conditions of thebinding treatment, and the like into consideration appropriately. Forexample, in cases where the terminally modifieddiacylphosphatidylethanolamine obtained by binding one terminus of adivalent reactive compound disuccinimidyl succinate to the terminalamino group of diacylphosphatidylethanolamine is used as the reactivephospholipid, 10 to 99% of the reactive phospholipid can be bound to thepeptide depending on selection of various conditions of the bindingtreatment. In such cases, the reactive phospholipid which is not boundto the peptide is comprised in the liposome as an acidic phospholipid.

Examples of a preferred mode of the peptide-bound liposome of thepresent invention include the following composition:

(I) 1 to 85 mol % of an acidic phospholipid comprising a C₁₄-C₂₄ acylgroup containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond;

(II) 0.01 to 80 mol % of a neutral phospholipid comprising a C₁₄-C₂₄acyl group containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbongroup containing one unsaturated bond;

(III) 0.2 to 80 mol % of a phospholipid, to which at least one peptideselected from the above-described peptide is bound, comprising a C₁₄-C₂₄acyl group containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbongroup containing one unsaturated bond; and

(IV) 0.2 to 75 mol % of a stabilizer.

(100 mol % in total)

Examples of a more preferred mode of the liposome to be used for thepeptide-bound liposome of the present invention include the followingcomposition:

the component (I) at a concentration of 2 to 80 mol %;

the component (II) at a concentration of 0.1 to 70 mol %;

the component (III) at a concentration of 0.3 to 60 mol %; and

the component (IV) at a concentration of 10 to 70 mol %.

(100 mol % in total)

Examples of a still more preferred mode of the liposome to be used forthe peptide-bound liposome of the present invention include thefollowing composition:

the component (I) at a concentration of 4 to 60 mol %;

the component (II) at a concentration of 0.1 to 60 mol %;

the component (III) at a concentration of 0.4 to 50 mol %; and

the component (IV) at a concentration of 20 to 60 mol %.

(100 mol % in total)

Examples of an especially preferred mode of the liposome to be used forthe peptide-bound liposome of the present invention include thefollowing composition:

the component (I) at a concentration of 5 to 40 mol %;

the component (II) at a concentration of 0.1 to 50 mol %;

the component (III) at a concentration of 0.5 to 25 mol %; and

the component (IV) at a concentration of 25 to 55 mol %.

(100 mol % in total)

The carbon number of the unsaturated acyl group or the unsaturatedhydrocarbon group to be comprised in the phospholipid in the liposomeused in the peptide-bound liposome of the present invention ischaracteristically 14 to 24, but a phospholipid comprising anunsaturated acyl group or unsaturated hydrocarbon group comprising acarbon number of less than 14 or more than 24 may also be comprised toan extent at which the effect of the present invention is not adverselyaffected. The ratio of the number of the C₁₄-C₂₄ unsaturated acyl groupor unsaturated hydrocarbon group with respect to the total number of allthe unsaturated acyl groups or unsaturated hydrocarbon groups comprisedin the phospholipid in the liposome used in the peptide-bound liposomeof the present invention is, for example, 50% or more, preferably 60% ormore, more preferably 75% or more, still more preferably 90% or more,most preferably 97% or more (for example, substantially 100%).

The liposome to be used for the peptide-bound liposome of the presentinvention may also comprise a lipid other than a phospholipid,comprising a C₁₄-C₂₄ acyl group or hydrocarbon group, as long as theeffect of the present invention is not adversely affected. The contentof the lipid is usually 40 mol % or less, preferably 20 mol % or less,more preferably 10 mol % or less, still more preferably 5 mol % or less(for example, substantially 0 mol %).

The liposome to be used in the present invention can be obtained by, forexample, a method wherein a phospholipid as a constituting component,reactive phospholipid, stabilizer, peptide and the like are used and areblended and processed in an appropriate way, followed by adding theresulting product to an appropriate solvent. Examples of the productionprocess include the extrusion method, vortex mixer method, ultrasonicmethod, surfactant removal method, reverse-phase evaporation method,ethanol injection method, prevesicle method, French press method, W/O/Wemulsion method, annealing method and freeze-thaw method. The particlediameter of the liposome is not restricted, however, in view of thestability during storage, the particle diameter is, for example, 20 to600 nm, preferably 30 to 500 nm, more preferably 40 to 400 nm, stillmore preferably 50 to 300 nm, most preferably 70 to 230 nm.

In the present invention, in order to enhance the physicochemicalstability of the liposome, sugars or polyols may be added to theinternal aqueous phase and/or the external aqueous phase during or afterpreparation of the liposome. In particular, in cases where the liposomeneeds to be stored for a long time or during formulation, a sugar or apolyol may be added/dissolved as a protective agent for the liposome,followed by removing water by freeze-drying to prepare a lyophilizedproduct of the phospholipid composition.

Examples of the sugars include monosaccharides such as glucose,galactose, mannose, fructose, inositol, ribose and xylose; disaccharidessuch as saccharose, lactose, cellobiose, trehalose and maltose;trisaccharides such as raffinose and melezitose; oligosaccharides suchas cyclodextrin; polysaccharides such as dextrin; and sugar alcoholssuch as xylitol, sorbitol, mannitol and maltitol. Among these sugars,monosaccharides and disaccharides are preferred, and glucose andsaccharose are especially preferred in view of availability and thelike.

Examples of the polyols include glycerin compounds such as glycerin,diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin,heptaglycerin, octaglycerin, nonaglycerin, decaglycerin andpolyglycerin; sugar alcohol compounds such as sorbitol and mannitol;ethylene glycol; diethylene glycol; triethylene glycol; tetraethyleneglycol; pentaethylene glycol; hexaethylene glycol; heptaethylene glycol;octaethylene glycol and nonaethylene glycol. Among these, glycerin,diglycerin, triglycerin, sorbitol, mannitol, and polyethylene glycolshaving molecular weights of 400 to 10,000 are preferred in view ofavailability. The concentration of the sugars or polyols to be containedin the internal aqueous phase and/or the external aqueous phase is, forexample, 1 to 20% by weight, preferably 2 to 10% by weight.

When the peptide-bound liposome of the present invention is produced,the peptide-bound liposome can be simply obtained by preparing aliposome to which the peptide has not been bound yet and then bindingthe peptide thereto. For example, a liposome which contains aphospholipid, stabilizer and a reactive phospholipid for binding thepeptide on the surface of the membrane is prepared as, for example, aliposome liquid, and sucrose, which is one of the sugars, is added toits external aqueous phase to about 2 to 10% by weight and dissolved.This sugar-added formulation is transferred to a 10 ml glass vial andthe vial is placed in a shelf freeze dryer, followed by cooling to, forexample, −40° C. to freeze the sample, thereby obtaining a lyophilizedproduct by a conventional method. The obtained lyophilized product canbe stored for a long time since water has been removed, and, whennecessary, by adding a particular peptide and carrying out the followingsteps, the peptide-bound liposome of the present invention can be simplyand rapidly obtained. In cases where the interaction between the peptideand the liposome is strong and strong instability is caused, it is avery simple way to store the liposome at the stage of a lyophilizedproduct like this and to use after binding the peptide when necessary.

The liposome to be used for the peptide-bound liposome of the presentinvention may comprise a phospholipid to which a peptide is bound.Examples of the method to obtain the liposome containing a phospholipidto which a peptide is bound include the methods by the following (A) and(B).

-   (A) A liposome containing a phospholipid, a reactive phospholipid    and a stabilizer is prepared, and a peptide and a divalent reactive    compound are added thereto, followed by linking a functional group    of the reactive phospholipid to a functional group of the peptide    via the divalent reactive compound. As the divalent reactive    compound, the one used for preparation of the terminally modified    derivatives of the reactive phospholipid may be similarly used.    Particular examples of the divalent reactive compound comprising an    aldehyde group include glyoxal, glutaraldehyde, succindialdehyde and    terephthalaldehyde. Preferred examples thereof include    glutaraldehyde. Examples of the divalent reactive compound    comprising a succinimide group include    dithiobis(succinimidylpropionate), ethylene    glycol-bis(succinimidylsuccinate), disuccinimidyl succinate,    disuccinimidyl suberate and disuccinimidyl glutarate. Furthermore,    examples of the divalent reactive compound comprising a succinimide    group at one terminus and a maleimide group at the other terminus    include N-succinimidyl-4-(p-maleimidophenyl)butyrate,    sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate,    N-succinimidyl-4-(p-maleimidophenyl)acetate,    N-succinimidyl-4-(p-maleimidophenyl)propionate,    succinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate,    sulfosuccinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate,    N-(γ-maleimidobutyryloxy)succinimide and    N-(ε-maleimidocaproyloxy)succinimide. By using such a divalent    reactive compound, a terminally modified derivatives of a reactive    phospholipid comprising a maleimide group as a functional group    (e.g., phosphatidylethanolamine) is obtained.-   (B) A method wherein a liposome containing a phospholipid, a    reactive phospholipid and a stabilizer is prepared, and a peptide is    added thereto, followed by linking a functional group of the    reactive phospholipid contained in the liposome to a functional    group of the peptide, and thereby binding the peptide to the    liposome.

Examples of the type of the bond in the above (A) and (B) include anionic bond, hydrophobic bond and covalent bond, and the type of the bondis preferably a covalent bond. Furthermore, particular examples of thecovalent bond include a Schiff base bond, amide bond, thioether bond andester bond. Both of the above two methods allow binding of the peptideto the reactive phospholipid contained in the liposome, thereby forminga phospholipid to which the peptide is bound in the liposome.

In the method (A), particular examples of the method to bind theliposome as a crude material to the peptide via a divalent reactivecompound include a method using a Schiff base bond. Examples of themethod to bind the liposome to the peptide via a Schiff base bondinclude a method wherein a liposome comprising an amino group on itssurface is prepared and the peptide is added to a suspension of theliposome, followed by adding dialdehyde as the divalent reactivecompound to the resulting mixture and binding the amino group on thesurface of the liposome to the amino group in the peptide via a Schiffbase.

Particular examples of this binding procedure include the followingmethod.

-   (A-1) In order to obtain a liposome comprising an amino group on its    surface, a reactive phospholipid comprising a C₁₄-C₂₄ acyl group    containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon group    containing one unsaturated bond (e.g., phosphatidylethanolamine) is    mixed with lipids as crude materials for the liposome (e.g., a    phospholipid and a stabilizer for a liposome), to prepare a liposome    wherein amino groups exist on the surface of the liposome in a    certain amount.-   (A-2) A peptide is added to the liposome suspension.-   (A-3) Subsequently, glutaraldehyde is added as the divalent reactive    compound, and the reaction is allowed to proceed for a certain    length of time, thereby allowing a Schiff base bond to be formed    between the liposome and the peptide.-   (A-4) Thereafter, in order to inactivate the reactivity of the    excess glutaraldehyde, glycine as an amino group-containing    water-soluble compound is added to the liposome suspension and    allowed to react therewith.-   (A-5) By a method such as gel filtration, dialysis, ultrafiltration    or centrifugation, the peptide unbound to the liposome, the reaction    product between glutaraldehyde and glycine, and excess glycine are    removed, to obtain a peptide-bound liposome suspension.

Particular examples of the method (B) include a method wherein areactive phospholipid comprising a functional group that can form anamide bond, thioether bond, Schiff base bond, ester bond or the like isintroduced to the phospholipid membrane. Particular examples of such afunctional base include succinimide, maleimide, amino, imino, carboxyl,hydroxyl and thiol. Examples of the reactive phospholipid to beintroduced to the liposome include the reactive phospholipid comprisinga C₁₄-C₂₄ acyl group containing one unsaturated bond, or a C₁₄-C₂₄hydrocarbon group containing one unsaturated bond (e.g.,phosphatidylethanolamine) whose amino terminus is modified.

A particular example of the binding procedure will now be describedreferring to a case in which diacylphosphatidylethanolamine is used.

-   (B-1) Diacylphosphatidylethanolamine comprising a C₁₄-C₂₄ acyl group    containing one unsaturated bond is reacted with disuccinimidyl    succinate by a known method at only one terminus, to obtain    disuccinimidyl succinate-bound diacylphosphatidylethanolamine    comprising a succinimide group as a functional group at the    terminus.-   (B-2) The disuccinimidyl succinate-bound    diacylphosphatidylethanolamine is mixed with other liposome    constituting components (e.g., a phospholipid and a stabilizer) by a    known method, to prepare a liposome composition comprising a    succinimide group on its surface as a functional group.-   (B-3) To the liposome composition suspension, a peptide is added,    and the amino group in the peptide is reacted with the succinimide    group on the surface of the phospholipid membrane.-   (B-4) The unreacted peptides, reaction byproducts and the like are    removed by a method such as gel filtration, dialysis,    ultrafiltration or centrifugation, to obtain a liposome suspension    containing a peptide-bound phospholipid.

In cases where a liposome is bound to a peptide, it is practicallypreferred to use an amino group or a thiol group, which is oftencomprised as a functional group. In cases where an amino group is used,a Schiff base bond can be formed by reacting it with a succinimidegroup. In cases where a thiol group is used, a thioether bond can beformed by reacting it with a maleimide group.

The antigen presenting cell in the present invention is a cell preparedby bringing a cell that expresses the cell surface antigen HLA-A2 intocontact with one or more types of peptides in vitro. The cell ispreferably autologous and/or allogenic. Examples of the cell includecells that express the cell surface antigen HLA-A2 (e.g., HLA-A*0201).The cell is preferably a lymphoid monocyte (T cell, macrophage, B cell,dendric cell or the like), more preferably a dendritic cell.

The antigen presenting cell of the present invention can be used as aninducer of HLA-A2-restricted CTLs specific to the SARS coronavirus,and/or as a vaccine for therapy or prophylaxis of infection of the SARScoronavirus, by administering (injecting) the cell to a subject. Thatis, the antigen presenting cell of the present invention presents thepeptide of the present invention on its surface and can strongly induceCTLs, so that the cell may be used as a CTL inducer and/or as a vaccinefor the purpose of elimination of the SARS coronavirus. The antigenpresenting cell is preferably prepared using a peptide at aconcentration of preferably 1 to 100 μM, more preferably 5 to 50 μM, forexample, 10 μM, per 10⁷ cells.

The peptide, the peptide-bound liposome and the antigen presenting cellof the present invention can be used as inducers of HLA-A2-restrictedCTLs specific to the SARS coronavirus, and/or as vaccines for therapy orprophylaxis of infection of the SARS coronavirus. The subject may be anyanimal including human. In a certain mode, the subject is human. Theinducer and/or vaccine of the present invention is/are made into acommon form as a pharmaceutical composition depending on the substanceregarded as the active ingredient, and direct delivery of thecomposition is generally achieved by parenteral injection (e.g.,subcutaneous injection, intraperitoneal injection, intravenousinjection, intramuscular injection, or injection to the space betweentissues). Examples of other administration methods include mucosaladministration (e.g., oral, transnasal or pulmonary), transocularadministration, percutaneous administration and administration bysuppositories.

That is, in cases where the composition is administered parenterally, itmay be administered in a dosage form such as an injection solution,transnasal agent, formulation for topical administration (e.g.,percutaneous preparation), or formulation for rectal administration. Incases where the composition is orally administered, it may beadministered in a dosage form usually used in the art. Examples of theinjection solution include sterile solutions or suspensions, andemulsions, and particular examples thereof include water,water-propylene glycol solutions, buffers and 0.4% physiological saline.Furthermore, in cases where the composition is made into a liquidformulation, it can be stored frozen, or stored after removing water byfreeze-drying or the like. When the freeze-dried formulation is to beused, it can be used by adding distilled water for injection or the likethereto and redissolving it. Examples of the formulation for topicaladministration include creams, ointments, lotions and percutaneouspreparations. Examples of the oral preparation or the formulation forrectal administration include capsules, tablets, pills, powders, drops,suppositories and liquids.

The above dosage forms are formulated by methods usually used in theart, together with pharmaceutically acceptable vehicles and additives.Examples of the pharmaceutically acceptable vehicles and additivesinclude carriers, binders, flavoring agents, buffering agents,thickening agents, coloring agents, stabilizers, emulsifiers,dispersants, suspending agents, antiseptics, pH adjusting agents,tonicity adjusting agents and wetting agents. Furthermore, examples ofthe pharmaceutically acceptable carriers include magnesium carbonate,lactose, pectin, starch and methyl cellulose.

The inducer of the present invention containing as an active ingredienta peptide, a peptide-bound liposome or an antigen presenting cell, andthe vaccine of the present invention containing as an active ingredienta peptide, a peptide-bound liposome or an antigen presenting cell mayfurther contain an adjuvant for enhancement of its effect. Examples ofthe adjuvant include aluminum hydroxide gel, Freund's complete adjuvant,Freund's incomplete adjuvant, pertussis adjuvant, poly(I,C) and CpG-DNA.Among these, CpG-DNA is preferred. CpG-DNA is a DNA containing anunmethylated CpG motif, and it can activate dendritic cells and enhancethe CTL induction by the peptide, peptide-bound liposome or antigenpresenting cell of the present invention.

The dose of the peptide of the present invention in the formulation, andthe number of doses of the formulation vary depending on the symptoms,age, body weight, dosage form and the like, and it is preferred toadminister usually 0.01 μg to 1 mg, preferably 0.1 μg to 500 μg, morepreferably 1.0 μg to 100 μg of the peptide once in every several days orseveral months. For example, for the primary immune response (that is,therapeutic or prophylactic administration), 1.0 μg to 500 μg of thepeptide is administered to an adult patient, and depending on theresponse and the conditions of the patient assayed by measurement of thespecific CTL activity in blood of the patient, boosting administrationof 1.0 μg to 100 μg of the peptide is subsequently carried out accordingto boosting therapy that continues for several weeks to several months.The number of the antigen presenting cells of the present invention tobe administered is preferably 10⁹ to 10⁶, more preferably 10⁸ to 10⁷,and the number of the cells may be appropriately controlled based on thesymptoms, age, body weight, dosage form and the like.

EXAMPLES

The present invention will now be described more concretely, but thepresent invention is not restricted to the Examples below.

Example 1 Prediction of CTL Epitopes

Using two kinds of computer software for prediction of epitopes, BIMAS<http://www-bimas.cit.nih.gov/molbio/hla_bind>) and SYFPEITHI(<http://www.syfpeithi.de>), epitope candidates for pp1a were searched.The search was carried out with the following settings: HLA Molecule,HLA-A*0201; and Peptide Length, 9 to 10 amino acid residues. Thirtykinds of epitope candidate peptides showing high prediction scores inthe both analytic methods were selected by the search. The amino acidsequences of these 30 kinds of epitope candidate peptides are shown inTable 1. For each of the 30 kinds of epitope candidate peptides, asynthetic peptide was prepared. Peptides that can actually function asepitopes were searched, and 9 kinds of epitopes were identified. Inorder to further analyze functions of the 9 kinds of peptides, theExamples 1 to 6 below were carried out.

TABLE 1 SEQ ID Epitope Sequence No: BIMAS SYFPEITHI  1) pp1a-15QLSLPVLQV 1 160.0 26  2) pp1a-103 TLGVLVPHV 2 160.0 26  3) pp1a-445TLNEDLLEI 3 98.4 28  4) pp1a-634 KLSAGVEFL 4 463.5 27  5) pp1a-651FLITGVFDI 5 640.2 27  6) pp1a-1121 ILLAPLLSA 6 71.9 26  7) pp1a-1139SLQVCVQTV 7 160.0 28  8) pp1a-1288 MLSRALKKV 8 272.0 25  9) pp1a-1652YLSSVLLAL 9 226.0 28 10) pp1a-2187 CLDAGINYV 10 351.9 27 11) pp1a-2207AMWLLLLSI 11 143.8 27 12) pp1a-2340 WLMWFIISI 12 1551.9 26 13) pp1a-2546ILLLDQVLV 13 437.5 26 14) pp1a-2754 TLLCVLAAL 14 181.8 29 15) pp1a-2755LLCVLAALV 15 118.2 25 16) pp1a-2758 VLAALVCYI 16 224.4 26 17) pp1a-2990ALSGVFCGV 17 132.1 25 18) pp1a-3444 VLAWLYAAV 18 177.4 27 19) pp1a-3459FLNRFTTTL 19 373.4 27 20) pp1a-3560 MLLTFLTSL 20 1174.4 29 21) pp1a-3564FLTSLLILV 21 735.9 25 22) pp1a-3616 FLLPSLATV 22 2722.7 33 23) pp1a-3687TLMNVITLV 23 591.9 25 24) pp1a-3709 SMWALVISV 24 958.9 28 25) pp1a-3730FLARAIVFV 25 4047.2 29 26) pp1a-3745 LLFITGNTL 26 134.4 26 27) pp1a-3816KLNIKLLGI 27 84.0 27 28) pp1a-3848 VLLSVLQQL 28 309.1 27 29) pp1a-4071ALWEIQQVV 29 970.0 25 30) pp1a-4219 VLGSLAATV 30 118.2 26

Example 2 Measurement of Binding Affinity of Peptide to HLA-A*0201Molecule

For the 30 kinds of epitope candidate peptides listed in Table 1, thebinding affinity to HLA-A*0201, which is a major histocompatibilitycomplex (MHC) class I molecule, was measured. The measurement wascarried out using T2 cells, which are human lymphoid cells. T2 cellslack the TAP gene and hence cannot transport self-peptides derived fromautoantigens. Therefore, these cells express HLA-A*0201, to whichpeptides are not bound, on the cell surfaces. When a peptide added tothe outside of the cell is bound to HLA-A*0201, an HLA-A*0201 complex isformed and becomes stable. Using this principle, the binding affinitywas calculated based on the relationship between the amount of theHLA-A*0201 complex formed and the concentration of the peptide added. Asthe antibody for detection of the HLA-A*0201 complex, an anti-HLA-A2monoclonal antibody BB7.2 (ATCC) was used. Furthermore, an epitope ofhepatitis C virus (NS3-1585) was used as a control.

More particularly, T2 cells were incubated at 37° C. overnight togetherwith various concentrations of peptides. Thereafter, the cells wereallowed to react with the anti-HLA-A2 antibody BB7.2, and then with aFITC-labeled secondary antibody, followed by analyzing the cells by flowcytometry. Using the mean fluorescence intensity (MFI) of the T2 cellspulsed with NS3-1585 as a standard (100%), the peptide concentration atwhich a mean fluorescence intensity of 50% was achieved was representedas BL₅₀ (half-maximal binding level) for each peptide and shown in Table2. BL₅₀ values of less than 100 μM, 100 to 200 μM, and more than 200 μMwere grouped into 3 categories “High”, “Medium” and “Low”, respectively,and 24 kinds of peptides showed high binding affinities.

TABLE 2 SEQ ID BL₅₀ Epitope No: (μM) Affinity  1) pp1a-15 1 75.7 High 2) pp1a-103 2 3.1 High  3) pp1a-445 3 19.2 High  4) pp1a-634 4 59.8High  5) pp1a-651 5 8.4 High  6) pp1a-1121 6 40.3 High  7) pp1a-1139 74.9 High  8) pp1a-1288 8 65.1 High  9) pp1a-1652 9 6.7 High 10)pp1a-2187 10 3.0 High 11) pp1a-2207 11 323.0 Low 12) pp1a-2340 12 2432.8Low 13) pp1a-2546 13 7.6 High 14) pp1a-2754 14 96.7 High 15) pp1a-275515 187.2 Medium 16) pp1a-2758 16 97.3 High 17) pp1a-2990 17 6.2 High 18)pp1a-3444 18 53.0 High 19) pp1a-3459 19 39.6 High 20) pp1a-3560 20 47.1High 21) pp1a-3564 21 100.4 Medium 22) pp1a-3616 22 31.8 High 23)pp1a-3687 23 22.8 High 24) pp1a-3709 24 6.4 High 25) pp1a-3730 25 25.7High 26) pp1a-3745 26 153.7 Medium 27) pp1a-3816 27 68.1 High 28)pp1a-3848 28 102.5 Medium 29) pp1a-4071 29 8.3 High 30) pp1a-4219 3053.3 High

Example 3 Induction of Peptide-Specific CTLs in Mice Immunized UsingAntigen Presenting Cells Pulsed with Peptides

In order to investigate whether or not CTLs are induced specifically tothe 30 kinds of epitope candidate peptides listed in Table 1, mice wereimmunized with mouse spleen cells pulsed with the peptides in vitro, andthe spleen cells were stimulated with the peptides followed by measuringthe CTL induction activities. As the mouse, the HLA-A2 transgenic mouse(HDD II mouse, Institut Pasteur, France; provided by Dr. F. Lemonnier)prepared by knocking out mouse MHC class I and β2-microglobulin (β2-m)in a mouse and introducing HLA-A*0201, which is a type of human MHCclass I, and the human β2-m gene was used. Using as an index the ratioof cells in which production of interferon-γ (IFN-γ) was promoted amongCD8-positive cells, the CTL induction activity was measured.

Spleen cells prepared from a naive HLA-A2 transgenic mouse wereincubated in vitro with each peptide at a concentration of 10 μM at 37°C. for hour. Another individual of a naive HLA-A2 transgenic mouse wasimmunized with the spleen cells pulsed with the peptide, by intravenousinjection.

One week after the immunization, spleen cells were prepared from theimmunized mouse, and the cells were suspended in a medium supplementedwith 10% fetal calf serum (FCS), followed by plating the cells in a96-well plate at the cell number of 2×10⁶ cells/well. To each well, eachpeptide (10 μM, final concentration) and 5 μL of 25-fold dilutedGOLGIPLUS™ solution (Japan BD) were added, and the resulting mixture wasincubated at 37° C. for 5 hours. Here, GOLGIPLUS™ was used to inhibitsecretion of produced IFN-γ by stopping intracellular transport. Afterwashing the cells, 1 μg/10⁶ cells of FcBlock antibody (Japan BD)suspended in 100 μL of FACS buffer (PBS containing 2% FCS and 0.1%sodium azide) was added to the cells and the resulting mixture wasincubated at 4° C. for 10 minutes in order to suppress nonspecificreaction by blocking the Fc receptors on the cell surfaces.

Subsequently, for detecting CD8-positive cells, cells were stained byadding 0.5 μg of a fluorescein isothiocyanate (FITC)-labeled CD8antibody to the cell suspension, and incubating the resulting mixture at4° C. for 30 minutes, followed by washing the cells twice. Furthermore,using intracellular cytokine staining (ICS), IFN-γ in the cells wasstained by the following procedure. First, 100 μL/well ofCYTOFIX/CYTOPERM™ solution (Japan BD) was added to the cells, and theresulting mixture was left to stand at 4° C. for 20 minutes to fix thecells and permeabilize the cell membrane. The cells were washed twice,and then collected. Subsequently, 0.5 μg of a phycoerythrin (PE)-labeledanti-IFN-γ antibody was added to the cells, and the cells were incubatedat 4° C. for 30 minutes. The cells were washed, and suspended in 100 μLof FACS fix buffer (PBS containing 2% FCS, 0.1% sodium azide and 1%formaldehyde), after which the cells were subjected to flow cytometryanalysis.

For each epitope candidate peptide, the ratio (%) of IFN-γ-positivecells among CD8-positive cells is shown in Table 3.

TABLE 3 SEQ ID ICS Epitope No: (% in CD8+ cells)  1) pp1a-15 1 0.05  2)pp1a-103 2 0.07  3) pp1a-445 3 0.08  4) pp1a-634 4 0.08  5) pp1a-651 50.02  6) pp1a-1121 6 0.05  7) pp1a-1139 7 0.07  8) pp1a-1288 8 0.05  9)pp1a-1652 9 0.05 10) pp1a-2187 10 *0.19 11) pp1a-2207 11 *0.48 12)pp1a-2340 12 *0.21 13) pp1a-2546 13 *0.17 14) pp1a-2754 14 0.04 15)pp1a-2755 15 *0.18 16) pp1a-2758 16 0.03 17) pp1a-2990 17 *0.16 18)pp1a-3444 18 *0.12 19) pp1a-3459 19 0.03 20) pp1a-3560 20 0.04 21)pp1a-3564 21 0.06 22) pp1a-3616 22 0.07 23) pp1a-3687 23 *0.20 24)pp1a-3709 24 *0.50 25) pp1a-3730 25 0.06 26) pp1a-3745 26 0.03 27)pp1a-3816 27 0.07 28) pp1a-3848 28 0.07 29) pp1a-4071 29 0.06 30)pp1a-4219 30 0.01 *ICS of 0.1% or more

As a result, among the 30 kinds of candidate peptides, 9 kinds ofpeptides (pp1a-2187, pp1a-2207, pp1a-2340, pp1a-2546, pp1a-2755,pp1a-2990, pp1a-3444, pp1a-3687 and pp1a-3709; SEQ ID NOs: 10, 11, 12,13, 15, 17, 18, 23 and 24) significantly induced IFN-γ-positive cellsamong CD8-positive cells, so that these 9 kinds of peptides weredetermined as the epitopes. These included peptides having low bindingaffinities to HLA-A*0201 (pp1a-2207 and pp1a-2340) and a peptide havinga medium binding affinity thereto (pp1a-2755), as shown in Example 2.

Example 4 Preparation of Liposomes and Peptide-Bound Liposomes

For the 9 kinds of CTL epitope peptides that showed especially high CTLinduction activities in Example 3 (pp1a-2187, pp1a-2207, pp1a-2340,pp1a-2546, pp1a-2755, pp1a-2990, pp1a-3444, pp1a-3687 and pp1a-3709; SEQID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24), peptide-bound liposomeswere prepared by the following method. Furthermore, in the same manner,liposomes to which a helper peptide (amino acid sequence: TPPAYRPPNAPIL;SEQ ID NO:32) was bound were prepared. As the helper peptide, onesynthesized based on the HBV core 128 helper peptide (OperonBiotechnologies) used by Dr. A. Sette et al. (e.g., Glenn Y et al., J.Immunol. 162:3915-3925 (1999)) was used.

(4-1) Synthesis of Reactive Phospholipid Composed of Terminally ModifiedPhosphatidylethanolamine (SuccinimidylGroup-Dioleoylphosphatidylethanolamine)

In 50 ml of chloroform, 2 g of dioleoylphosphatidylethanolamine and 180μl of triethylamine were dissolved/added, and the resulting solution wasplaced in a 300 ml four-necked flask. While stirring the solution in theflask with a magnetic stirrer at room temperature, a solution separatelyprepared by dissolving 3 g of disuccinimidyl suberate, which is adivalent reactive compound, in 80 ml of chloroform was added dropwise tothe solution according to a conventional method for 4 hours, therebyallowing one terminus of disuccinimidyl suberate to react with the aminogroup of dioleoylphosphatidylethanolamine. This crude reaction solutionwas transferred to an eggplant type flask, and the solvent wasevaporated with an evaporator. Subsequently, a small amount ofchloroform which is enough for dissolving the crude reaction product wasadded to the flask to obtain a high concentration crude reaction productsolution, and this solution was subjected to column chromatographyaccording to a conventional method using silica gel equilibrated withchloroform/methanol/water (65/25/1, volume ratio). Only the fraction ofinterest that contained dioleoylphosphatidylethanolamine whose aminogroup is bound to one terminus of disuccinimidyl suberate was recovered,and the solvent was evaporated, to obtain the succinimidylgroup-dioleoylphosphatidylethanolamine of interest.

(4-2) Preparation of Lipid Mixture Powder

In an eggplant type flask, 1.3354 g (1.6987 mmol) ofdioleoylphosphatidylcholine, 0.2886 g (0.2831 mmol) of succinimidylgroup-dioleoylphosphatidylethanolamine prepared in Example 4-1, 0.7663 g(1.9818 mmol) of cholesterol and 0.4513 g (0.5662 mmol) ofdioleoylphosphatidylglycerol sodium salt were placed, and 50 ml of achloroform/methanol/water (65/25/4, volume ratio)-mixed solvent wasadded thereto, followed by dissolving the reagents at 40° C.Subsequently, the solvent was evaporated under reduced pressure using arotary evaporator, to prepare a thin lipid membrane. Furthermore, 30 mlof distilled water for injection was added thereto, and the resultingmixture was stirred, to obtain a uniform slurry. This slurry was frozen,and dried in a freeze dryer for 24 hours, to obtain a lipid mixturepowder.

(4-3) Preparation of Liposomes

Subsequently, 60 ml of separately prepared buffer A (1.0 mMNa₂HPO₄/KH₂PO₄, 0.25 M saccharose, pH7.4) was placed in the eggplanttype flask containing the lipid mixture powder, and the lipids werehydrated while stirring the resulting mixture at 40° C., therebyliposomes were obtained. Thereafter, the particle diameters of theliposomes were adjusted using an extruder. The liposomes were firstallowed to pass through an 8-μm polycarbonate filter, and then through 5μm, 3 μm, 1 μm, 0.65 μm, 0.4 μm and 0.2 μm filters in this order. As aresult, liposome particles having an average particle diameter of 206 nm(measured by dynamic light scattering) were obtained.

(4-4) Preparation of Peptide-Bound Liposomes

In a 1.5 ml test tube, the liposomes obtained in Example 4-3 werecollected, and 3 ml of each peptide solution (1.25 mM)/buffer Aseparately prepared was added thereto, followed by stirring theresulting mixture at 5° C. for 48 hours to allow the reaction toproceed. This reaction liquid was subjected to gel filtration accordingto a conventional method using Sepharose CL-4B equilibrated with bufferA. Since the liposome fraction is turbid, the fraction of interest canbe easily recognized, but may also be confirmed using a UV detector orthe like. The phosphorus concentration of the thus obtained liposomesuspension was measured (Phospholipid Test, Wako), and the suspensionwas diluted with buffer A such that the concentration of phosphorusderived from the phospholipid is 2 mM, thereby obtaining a suspension ofeach peptide-bound liposome.

Example 5 Induction of Peptide-Specific CTLs in Mice Immunized UsingPeptide-Bound Liposomes

For the 9 kinds of CTL epitope peptides that showed especially high CTLinduction activities in Example 3 (pp1a-2187, pp1a-2207, pp1a-2340,pp1a-2546, pp1a-2755, pp1a-2990, pp1a-3444, pp1a-3687 and pp1a-3709; SEQID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24), the CTL inductionactivity in a mouse immunized using the peptide-bound liposome wasmeasured.

A naive HLA-A2 transgenic mouse was immunized at its foot pad with animmunization solution obtained by mixing the peptide-bound liposomes (25μl) and the helper peptide-bound liposomes prepared in Example 4 (25 μl)and an oligonucleic acid comprising a CpG motif (5 μg, base sequence:5′-tccatgacgt tctgatgtt-3′; SEQ ID NO:33) together. After theimmunization, the mouse was kept for 1 week, and the CTL inductionactivity was then measured by the same method as in Example 3. As acontrol, liposomes to which the peptide is not bound were used insteadof the peptide-bound liposomes. As the oligonucleic acid comprising aCpG motif, one prepared by gene synthesis (Hokkaido System Science Co.,Ltd.) based on Nagata. T. et al., Vaccine 25:4914-4921 (2007) was used.

Staining plots for CD8 and IFN-γ by flow cytometry in mice immunizedusing peptide-bound liposomes are shown in FIG. 1. Each dot inside theboxed area in the upper right of each graph corresponds to anIFN-γ-positive cell among CD8-positive cells, and the value shown in theboxed area indicates the ratio (%) of the IFN-γ-positive cells among theCD8-positive cells. As a result, it was revealed that immunization withthe contained peptide-bound liposomes causes CTL induction.

Comparative Example

As a comparative control experiment for Example 5, the followingexperiment was carried out. Measurement of the CTL induction activitywas carried out by the same method as in Example 4 except that, insteadof a CTL epitope peptide of pp1a, a CTL epitope peptide of the Spikeprotein of SARS-CoV (spike-1203, amino acid sequence: FIAGLIAIV; SEQ IDNO:34) was used and that a naive HLA-A2 transgenic mouse was immunizedby intramuscular injection at thigh muscle.

Staining plots for CD8 and IFN-γ by flow cytometry in mice immunizedusing the spike-1203-peptide-bound liposomes are shown in FIG. 2. As aresult of the analysis, the ratio (%) of IFN-γ-positive cells amongCD8-positive cells was 0.29 when the known CTL epitope peptide of theSpike protein (spike-1203) was used. When compared to the results inExample 4, it was revealed that 8 kinds of CTL epitope peptides derivedfrom pp1a (pp1a-2187, pp1a-2207, pp1a-2340, pp1a-2546, pp1a-2755,pp1a-2990, pp1a-3687 and pp1a-3709; SEQ ID NOs: 10, 11, 12, 13, 15, 17,23 and 24) show CTL induction activities higher than that of the CTLepitope peptide of the Spike protein.

Example 6 In Vivo CTL assay in Mice Immunized Using Peptide-BoundLiposomes

For 5 kinds of CTL epitope peptides that showed high CTL inductionactivities in Example 4 (pp1a-2187, pp1a-2340, pp1a-2755, pp1a-2990 andpp1a-3709; SEQ ID NOs: 10, 12, 15, 17 and 24), the CTL response activityin a mouse immunized using the peptide-bound liposome was measured invivo.

The principle of measurement of the CTL response activity in vivo was asfollows. Target cells which were not pulsed with the peptide (negativecontrol) were labeled with carboxy fluorescein diacetate succinimidylester (CFSE) at a low concentration, and, on the other hand, targetcells pulsed with the peptide were labeled with CFSE at a highconcentration (10-fold). The same number of cells of the 2 kinds of cellpopulations were mixed together, and transferred to a mousepreliminarily immunized using the peptide-bound liposomes. Thereafter,spleen cells were collected from the mouse to which the mixed cells weretransferred, and a spleen cell suspension was prepared, followed bymeasuring the ratio of the transferred cells labeled with CFSE by flowcytometry analysis. In a mouse having no CTL response activity to theantigen peptide, equal amounts of the CFSE-labeled transferred cells arecollected. On the other hand, since, in a mouse having the CTL responseactivity to the antigen peptide, the target cells covered with theantigen peptide are lysed, the degree of the lysis in vivo can bemeasured by quantifying decrease in the cells highly labeled with CFSEby flow cytometry.

More particularly, the measurement was carried out by the followingmethod.

A mouse for the transfer was preliminarily prepared by immunizing amouse with the peptide-bound liposomes prepared using each peptide andinducing the peptide-specific CTLs. As a control, a mouse immunized withliposomes to which the peptide was not bound, instead of thepeptide-bound liposomes, was used. One week after the immunization,spleen cells were prepared from another individual of the naive mouse,and the cells were suspended in RPMI-1640 (Sigma) at the cell number ofthe medium is 2×10⁸ cells/2 mL. A 1 mL aliquot of the resultingsuspension was taken into each of two tubes. To only one of the tubes,the peptide was added to a final concentration of 10 μM, and the twotubes were incubated at 37° C. for 1 to 2 hours. After washing the cellsonce, the cells were suspended in 20 mL of PBS/0.1% BSA and then brieflyvortexed. Thereafter, to the tube containing the cells pulsed with thepeptide, 10 μL of 5 mM CFSE was added, immediately followed by adding 1μL of 5 mM CFSE to the tube containing the cells which were notincubated with the peptide. The both tubes were vortexed to suspend thecells, and incubated in a water bath at 37° C. for 10 minutes. The bothcell populations were centrifuged and washed once, followed by countingthe live cell numbers and suspending each cell population in HBSS to aconcentration of 5×10⁷ cells/mL. Equal amounts of the cell suspensionswere mixed together to prepare an immunization liquid, and 200 μL (1×10⁷cells/individual) of the resulting cell suspension was intravenouslyinjected to a mouse preliminarily immunized, thereby carrying out thecell transfer. Twelve hours after the transfer, spleen cells werecollected from the mouse, and suspended in 1 mL/spleen of PBS/1% FBS/5mM EDTA. The cells were centrifuged and suspended in 1 mL of FACS fixbuffer, and 100 μL of this spleen cell suspension was diluted with 2 mLof FACS buffer, followed by subjecting the resulting dilution to flowcytometry analysis.

Staining plots for CFSE by flow cytometry in mice to which the targetcells labeled with CFSE were transferred are shown in FIG. 3. Therightmost peak in each graph indicates the cell population pulsed withthe peptide, and the peak in its left indicates the cells which were notpulsed with the peptide. It was revealed that, in each CTL epitopepeptide, the mouse immunized with the peptide-bound liposomes (rightgraph) shows a decreased number of the cells pulsed with the peptidecompared to the control immunized with the liposomes (left graph). Thepeptide-specific lysis rates (%) of the target cells by CTLs were:pp1a-2187, 79.2%; pp1a-2340, 68.5%; pp1a-2755, 70.5%; pp1a-2990, 73.4%;and pp1a-3709, 96.3%; showing that all of these peptides have high CTLresponse activities. Among the peptides, pp1a-3709 was revealed to havean especially high CTL response activity. Therefore, it was revealedthat these CTL epitope peptides (pp1a-2187, pp1a-2340, pp1a-2755,pp1a-2990 and pp1a-3709; SEQ ID NOs: 10, 12, 15, 17 and 24) are dominantpeptides having high CTL response activities.

The invention claimed is:
 1. A peptide-bound liposome, wherein thepeptide is covalently bound to the surface of the liposome; the liposomecomprises: a phospholipid comprising a C₁₄-C₂₄ acyl group containing oneunsaturated bond, or a C₁₄-C₂₄ hydrocarbon group containing oneunsaturated bond, and a stabilizer; and the peptide comprises the aminoacid sequence of SEQ ID NO: 24, wherein the peptide-bound liposomeinduces an immune response that produces human leukocyte antigen type A2(HLA-A2)-restricted cytotoxic T lymphocytes (CTLs) specific to severeacute respiratory syndrome (SARS) coronavirus.
 2. The peptide-boundliposome according to claim 1, wherein the phospholipid comprises aC₁₄-C₂₄ acyl group containing one unsaturated bond.
 3. The peptide-boundliposome according to claim 1, wherein the phospholipid comprises anoleoyl group.
 4. The peptide-bound liposome according to claim 1,wherein the phospholipid is at least one selected from the groupconsisting of diacylphosphatidylserine, diacylphosphatidylglycerol,diacylphosphatidic acid, diacylphosphatidylcholine,diacylphosphatidylethanolamine,succinimidyl-diacylphosphatidylethanolamine andmaleimide-diacylphosphatidylethanolamine.
 5. The peptide-bound liposomeaccording to claim 1, wherein the stabilizer is cholesterol.
 6. Thepeptide-bound liposome according to claim 1, wherein the peptide iscovalently bound to the phospholipid on the surface of the liposome. 7.The peptide-bound liposome according to claim 1, wherein the liposomecomprises: (A) 1 to 99.8 mol % of a phospholipid comprising: a C₁₄-C₂₄acyl group containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbongroup containing one unsaturated bond; and (B) 0.2 to 75 mol % of astabilizer.
 8. A peptide-bound liposome according to claim 1 comprising:(I) 1 to 85 mol % of an acidic phospholipid comprising a C₁₄-C₂₄ acylgroup containing one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond; (II) 0.01 to 80 mol % of a neutralphospholipid comprising a C₁₄-C₂₄ acyl group containing one unsaturatedbond, or a C₁₄-C₂₄ hydrocarbon group containing one unsaturated bond;(III) 0.2 to 80 mol % of a phospholipid comprising a C₁₄-C₂₄ acyl groupcontaining one unsaturated bond, or a C₁₄-C₂₄ hydrocarbon groupcontaining one unsaturated bond, wherein the phospholipid is bound tothe at least one peptide; and (IV) 0.2 to 75 mol % of a stabilizer. 9.An inducing agent for human leukocyte antigen type A2(HLA-A2)-restricted cytotoxic T lymphocytes (CTLs) specific to severeacute respiratory syndrome (SARS) coronavirus comprising: thepeptide-bound liposome according to claim 1 as an active ingredient, anda pharmaceutically acceptable carrier, wherein the agent induces animmune response that produces HLA-A2-restricted CTLs specific to SARScoronavirus.
 10. A vaccine comprising: the peptide-bound liposomeaccording to claim 1, and a pharmaceutically acceptable carrier, whereinthe vaccine induces an immune response that produces HLA-A2-restrictedCTLs specific to SARS coronavirus.
 11. The inducing agent forHLA-A2-restricted CTLs specific to the SARS coronavirus according toclaim 9, further comprising CpG-DNA.
 12. The vaccine according to claim10, further comprising CpG-DNA.
 13. A method of producing an immuneresponse to severe acute respiratory syndrome (SARS) coronavirus in asubject, comprising administering to the subject an effective amount ofthe vaccine composition of claim 10, thereby inducing the immuneresponse to SARS coronavirus in the subject.
 14. The method of claim 13,wherein the subject is human.